Stably transfected cell line brought to my lab was put in quaratine. Cultured without antibiotics for over 6 weeks, grows great, no obvious problems. Samples were send to a GLP facility for Mycoplasma tests.3 separate PCR tests came back positive (they do DNA extraction, use their own proprietory primers), additional PCR done with Sigma Mycoplasma detection kit was also positive in sample containing cells, NEGATIVE from supernatant.2 separate samples (the same culture) came back clear NEGATIVE from Hoechst stain test using VERO indicator cells. The second sample was from culture already 4 weeks without antibiotics.

I am not very versed in transfection - but is it possible that when these cells were transfected and might have been mycoplasma positive, the mycoplasma DNA got incorporated into the cell DNA as well?The mycoplasma infection itself might have been wiped out by special treatment since, but the DNA trace is there, although there is no evidence of active Mycoplasma in these cells.Have you ever heard of anything like this?

Hmmh, I'm not sure if I got your situation right, but it is possible that sensitive DNA-based methods detect mycoplasma DNA from samples even if there aren't live mycoplasma around any more. Hoechst & Vero should be good to show mycolpasma, so I'm a bit puzzled by your results as well, especially that Sigma kit negative/positive result. I've never heard that someone would've mistakenly incorporated mycoplasma DNA into host cells, which could after that keep growing and retain the myco DNA. And since mycoplasma DNA doesn't transfect eukaryotic DNA by natural means...

...my best guess still is that there is some residual mycoplasma DNA among the cultures, but maybe no live mycoplasma. Your best way to be sure is to grow them antibiotic free and keep testing.

Thank you, wouldn't it be great if there is a 100% reliable test?:)Now I am looking into sending the sample to direct broth culture test and also hoping to find a lab that would do NASBA test for Mycoplasma mRNA - to try to distinguish between residual Myco DNA and actual very low live Myco infection.Question: That residual Myco DNA... how long can it persist in cell cultures? There clearly is no live active Mycoplasma, it would have shown up by now. These cells have been grown and paggased extensively without antibiotics for 2 months. Where in the cell would this residual DNA be sequestered?

About residual DNA - I don't know. Even though DNA is a relatively stable molecule compared to many other biomolecules, I find it hard to believe that even PCR could detect it after two months of culture and passages. There might be some other source of contamination, though, like some reagent or equipment that is kept in cold/ice and that is used in your cultures. Maybe it contains no more live mycoplasma (mycoplasma die easily outside their favourite environment), but traces of DNA could remain there.

The Mycoplasma DNA can remain in the cultures for over 6 weeks therefore PCR based detection picks up infection even if its dead. the other tests require live Mycoplasma hence your is it isn't it dilema